Browse and James [19], in 1964, published the first cohort of PE patients submitted to thrombolysis with streptokinase. The first case, a 56-year-old man with history of recurrent deep vein thrombosis who was under long-term (9 month) anticoagulation treatment with phenindione and stopped treatment 18 months before of the new acute PE event. A month before admission he had clinical signs of left deep venous thrombosis, breathlessness, and palpitations. Three days before admission breathlessness worsened and he had syncope. When admitted in emergency room with severe chest pain, clinical examination showed respiratory failure, jugular distention, hypotension (80/75 mmHg), and ankle edema. Electrocardiogram had T wave inversion in leads V₁, V₂, and V₃. Clinical diagnosis of PE was established and treatment with heparin, oxygen, and vasopressors was started. On the fourth day, the clinical conditions worsened despite treatment with heparin. On electrocardiogram, right ventricular strain was identified. Streptokinase was given on the fifth, sixth, and seventh days in 8-h intravenous infusion of two mega units each. A maintenance dose about 70,000 units per hour to maintain a reasonably high level of fibrinolytic activity was considered. As adjunctive treatment prednisone 15 mg daily and 10,000 units of heparin was given intravenously between the infusions and during 2 days after treatment. Given the history of recurrent deep venous thrombosis, long-term anticoagulation with phenindione was considered. On the sixth day, blood pressure returned to normal, however the dyspnea was worse and chest pain remained. Twenty-four hours after he has been kept on phenindione, and had good outcome.

This case exemplifies how long-term streptokinase infusion was successful in a massive PE patient. Previous experience in streptokinase development allowed to establish dose and time infusion.

Streptokinase was the first thrombolytic developed for therapeutic use in the United States, using material purified from culture filtrates of the H46A isolate of Streptococcus equisimilis (Lancefield Group C). All commercial streptokinase currently available for human use is derived from this strain [1]. In the 1960s, European pharmaceutical companies conducted the first clinical trial in humans [20]. At this time, the Committee on Biological Standardization of the World Health Organization identified the need for international standards (IS) for streptokinase potency measurement. Four years later the international unit (IU) for this fibrinolytic was defined as an activity contained in 0.002090 mg of the first IS [1].

Streptokinase is a nonenzyme protein with molecular weight of 47,000 Da produced by β-hemolytic streptococci, which indirectly activates the fibrinolytic system. The drug forms a 1:1 stoichiometric complex with plasminogen which exposes and activates a site in the modified plasminogen moiety, whereby the complex becomes a potent plasminogen activator. The main difference with fibrin-specific fibrinolytics is that streptokinase causes an indirect conformational change in the plasminogen molecule, which then acts as plasmin [1].

Currently, streptokinase remains as the most widely used thrombolytic agent and it is now produced in many developing countries, being an attractive therapeutic option because of its low cost. As a bacterial product, it is immunogenic, which may reduce the effectiveness, and/or cause allergic reactions. Other agents (fibrin-specific) are now generally more used in developed nations. The quality of streptokinase in Europe is maintained and well regulated; however, this could not be the same in developing countries [1] possibly with large discrepancies in activity, purity, and composition. Recently, a wide range of streptokinase products used in India, South Korea, South America, and the Middle East were monitored. The main observation was the poor quality control used to treat ST-elevation myocardial infarction. In this result, deterioration of product during transport was ruled out [21]. Another point to consider is whether patients surviving treatment with suboptimal streptokinase dose will develop antibodies compromising future treatments or poor outcome; this remains as an open question.

Another major source of concern is the standardization of recombinant streptokinase. Several companies are producing recombinant streptokinase engineered in an attempt to reduce immunogenicity, and others are developing new variants of this agent to improve fibrin-specificity. It is known that small changes in the sequence of native streptokinase at the amino and carboxy terminals can significantly impact the activity and fibrin-dependence. Streptokinase in short-dose and long-term peripheral vein infusion have been approved by FDA for the treatment of massive PE patients (Table 2.2). Table shows FDA-approved and non-approved thrombolytic regimens. In the contemporary era, streptokinase in 1,500,000 IU short-term one-hour infusion by peripheral vein was the first regimen that improved in-hospital mortality in severe right ventricular dysfunction PE patients in a controlled-randomized trial [22].

In 1985, Bounameaux and colleagues [36] published the first successful case with rt-PA in a 63-year-old man with angiographically proved massive PE, hospitalized one-hour after sudden onset of severe dyspnea; patient had a history of renal transplant 5 weeks before onset PE symptoms. On admission, he was cyanotic, polypneic, with blood pressure 145/90 mmHg and heart rate 117/min., and without signs of deep venous thrombosis. Arterial blood gases showed severe hypoxia and hypocapnia. Electrocardiogram was normal, chest X-ray showed atelectasis of the upper lobe of right lung and sequelae of tuberculosis and pulmonary angiography with massive bilateral obstruction of the main pulmonary artery. Non-fractionated heparin infusion was started at the dosage of 30,000 IU/24 h.

rt-PA infusion was started 8 h after onset symptoms through a catheter in right ventricle at a dose of 0.5 mg/kg body weight in 90 min until completing 30 mg. Thirty minutes later patient clinically improved with a progressive increase in arterial PO₂. Pulmonary angiography 24 h after the infusion showed almost complete recanalization of the branch to the right inferior lobe. Patient has transient atrial flutter successfully treated with digital on the second day after thrombolysis. No bleeding events, neither further complication occurred.

The first report of successful thrombolysis using rt-PA confirmed the efficacy of this agent in the setting of angiographic massive PE. Although the infusion was driven by direct catheter in right ventricle, previous clinical experience in ST-elevation myocardial infarction patients was considered. Additionally, this case exemplifies the usefulness of an rt-PA reduced dose as a therapeutic approach in pulmonary artery thrombus.

This activator is a serine protease in which His 322, Asp 371, and Ser 478 constitute the active site residues; rt-PA is blocked by the active site serine inhibitors diisopropylfluorophosphate (DFP) [37, 38] and chloromethyl ketones, which inhibit the active site histidine. Although Tos-Lys-CH₂Cl (TLCK) weakly inhibits rt-PA, d-Phe-Pro-Arg-CH₂Cl is relatively a strong rt-PA inhibitor [39]. Both one-chain rt-PA and two-chain rt-PA react with DFP. A useful, commercially available tripeptide substrate is H-d-Ile-Pro-Arg-pNA; which is, however, not specific for rt-PA but sensitive to a broad spectrum of serine proteases. Two-chain rt-PA is more active towards low-molecular-weight-substrates [40].

Recombinant human tissue-type plasminogen activator has a specific affinity for fibrin [41]. When rt-PA is present during formation of fibrin clots of increasing density, half-maximal binding occurred at approximately 0–14 g per 1 (0.4 μM) [42]. Experiments on plasminogen activation in the presence of varying amounts of fibrin suggest a dissociation constant of 0.14 μM [30]. Detailed binding studies, however are still lacking, as is the localization of the binding sites, both in the fibrin molecule and in rt-PA.

Recombinant human tissue-type plasminogen activator is a poor enzyme in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen [43, 44]. This has been explained by an increased affinity of fibrin-bound rt-PA for plasminogen without significantly influencing the catalytic efficiency of the enzyme [30]. The kinetic data of Hoylaerts et al. [30] support a mechanism in which rt-PA and plasminogen adsorb to a fibrin clot in a sequential and ordered way, yielding a ternary complex. Fibrin essentially increases the local plasminogen concentration by creating an additional interaction between rt-PA and its substrate. The high affinity of rt-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, although no efficient plasminogen activation by rt-PA occurs in plasma. In another kinetic study Randby [45] observed significantly increased turnover in the presence of fibrin.

Recombinant human tissue-type plasminogen activator-mediated plasminogen activation is also potentiated by fibrin monomer and by cyanogen bromide (CNBr)-digested fibrinogen [46], suggesting that the polymeric fibrin structure is not a prerequisite for the stimulation. It has been reported that the potentiating activity of CNBr-digested fibrinogen resides in the CNBr fragment FCB-2 (Hol-DSK) and more particularly in the fragment consisting of residues 148–207 of the α chain [47]. Stimulation of the activation of plasminogen by rt-PA has also been reported in the presence of denatured proteins, such as fibrinogen, IgG, and ovalbumin [48, 49], and it has been speculated that the ability of rt-PA to recognize misfolded proteins indicates a role in selective catabolism of damaged protein, in general, not solely fibrin clots [48].

A remarkable difference in K _M values in the presence of fibrin was found in two studies from the same laboratory. Hoylaerts et al. [30], using an assay system in which fibrin could be degraded during the experiment, found a K _M of 0.16 μM, whereas Rijken et al. [42], using a system in which lysis of fibrin was inhibited by an excess of aprotinin, found a K _M of 1.1 μM. The difference may be explained by recent results of Suenson et al. [49], who disclosed a strong plasminogen-binding site upon initial fibrin degradation. A K _M value of 1.1 μM [42], close to the physiological plasminogen concentration in plasma, may indicate a regulatory role for histidine-rich glycoprotein, which interacts with plasminogen and thus influences the free plasminogen concentration [50].

Recombinant human tissue-type plasminogen activator occurs either as a one-chain molecule or as a proteolytically degraded two-chain molecule. In contrast to analogous enzyme system, single-chain rt-PA is, however, not an inactive precursor but an active enzyme. Although the single-chain form is less active towards low-molecular-weight-substrates and inhibitors [40], the two forms are almost equally active towards plasminogen [42, 45].

Functional domains responsible for the fibrin-binding and for the catalytic activity of rt-PA have been localized in the rt-PA molecule [51, 52]. Holvoet et al. [51] partially reduced two-chain rt-PA and separated the A chain and the B chain by immune adsorption on monoclonal antibodies reacting with the fibrin-binding site and with the active center of the enzyme, respectively. The purified B chain activated plasminogen following Michaelis-Menten kinetics, with kinetic constants similar to those of intact rt-PA, but fibrin did not stimulate the activation of plasminogen by the B chain. The purified A chain bound to fibrin with an affinity similar to that of intact rt-PA but did not activate plasminogen.

Plasmin, the proteolytic enzyme of the fibrinolytic system, is a serine protease with relatively low substrate specificity. In purified systems, it will degrade fibrinogen as well as fibrin. When plasmin circulates freely in the blood, it will degrade a number of plasma proteins, including fibrinogen and the blood coagulation factors V and VIII.